The biosynthesis and metabolism of amino acids is of critical importance in many metabolic and catabolic pathways in cells, and is fundamental to the production of cellular proteins. A wide array of enzymes facilitate the synthesis, interconversion, and degradation of amino acids, including transaminases, oxidases, reductases, dehydrogenases, and kinases, among many others. One such family of enzymes, the serine and threonine dehydratases, catalyze the irreversible deamination of serine or threonine to pyruvate or 2-oxobutyrate, respectively.
The reaction mechanism for these enzymes has been characterized (Snell and Di Mari (1970) The Enzymes (Boyer, P. D., ed.), Academic Press: 3rd ed. Vol. 2: 335-370; and Ogawa et al. (1989) Biochim. Biophys. Acta 996: 139-141). First, a Schiff base is formed between a pyridoxal-5xe2x80x2phosphate cofactor and a specific lysine residue which is strictly conserved within the serine and threonine dehydratase family. A new Schiff base is subsequently formed between the cofactor and the hydroxyamino acid by transimination, catalyzing the removal of the xcex1-proton through stabilization of the resulting carbanion by the planar xcfx80-system of the prosthetic group. The hydroxyl group is eliminated, and the resultant enamine is freed by a second transimination. A tautomerization step results in the formation of a ketimine, which hydrolyses to the 2-oxoacid and ammonia (Gabowski et al. (1993) Trends in Biological Sciences 18: 297-300). A subclass of the serine dehydratases found in anaerobic bacteria substitutes an iron-sulfur cofactor for pyridoxal-5xe2x80x2-phosphate, and exhibits an altered reaction mechanism with similarities to the mechanism of aconitase (Hofmeister et al. (1993) Eur J Biochem 215(2):341-9). Threonine dehydratases, in general, are able to deaminate either threonine or serine, while the serine dehydratases have been found to be specific for the deamination of serine (Grabowski et al. (1992) Eur. J. Biochem. 199:89-94; and Alfoldi et al. (1968) J. Bacteriol. 96:1512-1518).
Members of the serine and threonine dehydratase family are found in nearly all organisms, from bacteria to yeast to mammals. Alignments of the amino acid sequences of family members from disparate organisms have revealed two conserved regions, termed C1 and C2. The conserved C1 domain is located approximately 50 amino acid residues from the N-terminus of the enzyme, and includes the consensus sequence (G)S(F)K(I)RG (Datta et al. (1987) Proc. Natl. Acad. Sci. USA 84: 393-397). This region of the protein has been shown to bind the cofactor, pyridoxal-5xe2x80x2-phosphate, at the conserved lysine residue (Schlitz and Schmitt (1981) FEBS Lett. 134:57-62). Conserved region C2 is located in the central region of the amino acid sequences of these enzymes, and is predicted to have a beta sheet-coil-beta sheet structure (Datta et al., supra). C2 is rich in glycine, and is thought to be involved in the catalytic activity of the enzymes (Marceau et al. (1988) J. Biol. Chem. 263: 16926-16933).
Serine and threonine dehydratases play key roles in the degradation of threonine and serine, as well as in the biosynthesis of isoleucine and the production of pyruvate and 2-oxobutyrate, both of which serve as substrates for energy metabolism or biosynthetic purposes. As such, the activity of these dehydratases contributes to the ability of the cell to grow and differentiate, to proliferate, and to communicate and interact with other cells (for example, through the production of growth factors and cytokines).
The present invention is based, at least in part, on the discovery of novel members of the family of dehydratase molecules, referred to herein as DHY nucleic acid and protein molecules. The DHY nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cellular proliferation, growth, differentiation, or migration. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding DHY proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of DHY-encoding nucleic acids.
In one embodiment, a DHY nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1 or 3, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-106 of SEQ ID NO:1. In yet a further embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1097-1327 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:1 or 3.
In another embodiment, a DHY nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, a DHY nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the amino acid sequence of SEQ ID NO:2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human DHY. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2. In yet another preferred embodiment, the nucleic acid molecule is at leas 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 950-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300 or more nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300 or more nucleotides in length and encodes a protein having a DHY activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably DHY nucleic acid molecules, which specifically detect DHY nucleic acid molecules relative to nucleic acid molecules encoding non-DHY proteins. For example, in one embodiment, such a nucleic acid molecule is at least 20-30, 3040,40-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700,700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or a complement thereof.
In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., 15 contiguous) nucleotides in length and hybridize under stringent conditions to the nucleotide molecule set forth in SEQ ID NO:1.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or 3, respectively, under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a DHY nucleic acid molecule, e.g., the coding strand of a DHY nucleic acid molecule.
Another aspect of the invention provides a vector comprising a DHY nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably a DHY protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant DHY proteins and polypeptides. In one embodiment, an isolated DHY protein includes at least one or more of the following domains: a transmembrane domain, a serine/threonine dehydratase pyridoxal-phosphate attachment site, a serine/threonine dehydratase domain, and/or pyridoxal phosphate-dependent lyase synthase domain.
In a preferred embodiment, a DHY protein includes at least one or more of the following domains: a transmembrane domain, a serine/threonine debydratase pyridoxal-phosphate attachment site, a serine/threonine dehydratase domain, and/or a pyridoxal phosphate-dependent lyase synthase domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, a DHY protein includes at least one or more of the following domains: a transmembrane domain, a serine/threonine dehydratase pyridoxal-phosphate attachment site, a serine/threonine dehydratase domain, and/or a pyridoxal phosphate-dependent lyase synthase domain, and has a DHY activity (as described herein).
In yet another preferred embodiment, a DHY protein includes at least one or more of the following domains: a transmembrane domain, a serine/threonine dehydratase pyridoxal-phosphate attachment site, a serine/threonine dehydratase domain, and/or a pyridoxal phosphate-dependent lyase synthase domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3.
In another embodiment, the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2. In another embodiment, a DHY protein has the amino acid sequence of SEQ ID NO:2.
In another embodiment, the invention features a DHY protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof. This invention further features a DHY protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof.
The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-DHY polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably DHY proteins. In addition, the DHY proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the presence of a DHY nucleic acid molecule, protein, or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a DHY nucleic acid molecule, protein, or polypeptide such that the presence of a DHY nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of DHY activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of DHY activity such that the presence of DHY activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating DHY activity comprising contacting a cell capable of expressing DHY with an agent that modulates DHY activity such that DHY activity in the cell is modulated. In one embodiment, the agent inhibits DHY activity. In another embodiment, the agent stimulates DHY activity. In one embodiment, the agent is an antibody that specifically binds to a DHY protein. In another embodiment, the agent modulates expression of DHY by modulating transcription of a DHY gene or translation of a DHY mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a DHY mRNA or a DHY gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted DHY protein or nucleic acid expression or activity by administering an agent which is a DHY modulator to the subject. In one embodiment, the DHY modulator is a DHY protein. In another embodiment the DHY modulator is a DHY nucleic acid molecule. In yet another embodiment, the DHY modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant or unwanted DHY protein or nucleic acid expression is a dehydratase-associated disorder, e.g., a CNS disorder, a cardiovascular disorder, a muscular disorder, or a cell proliferation, growth, differentiation, or migration disorder.
The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a DHY protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a DHY protein, wherein a wild-type form of the gene encodes a protein with a DHY activity.
In another aspect the invention provides methods for identifying a compound that binds to or modulates the activity of a DHY protein, by providing an indicator composition comprising a DHY protein having DHY activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on DHY activity in the indicator composition to identify a compound that modulates the activity of a DHY protein.